Known immunoassays include, for example, both the classical radio-immunoassay in which the antigen is labelled and the immunoradiometric assay in which labelled antibodies are used. The principles that govern the competitive binding of ligands to specific receptors are pertinent to the classical radio-immunoassay. The antibody, produced in response to an antigen, serves as the receptor. The ligand in the test sample competes with a constant amount of labelled ligand (i.e. the antigen or suitable derivative) for a limited number of combining sites on the antibody molecules. After equilibration, free labelled antigen is separated from antibody-bound labelled antigen and the radio-activity present in the free or bound state is measured. The extent of the competition between the labelled and unlabelled ligand for the antibody is compared to the inhibitory effectiveness observed with known concentrations of standards. A different type of immunoassay is the immunoradiometric assay (IRMA), based on the use of labelled specific antibodies. In an assay, standards or unknown samples are incubated with an excess of labelled antibody and, after equilibrium has been attained, the unused labelled antibody is removed and the bound fraction counted. In addition to conventional IRMA, "sandwich" techniques have been developed for the assay or large molecules with more than one antigenic determinant. In this case, the standard or sample is first incubated with an excess of antibody bound to an insoluble support and directed against one antigenic determinant. After careful washing, excess of a second, labelled antibody directed against another part of the molecule is added. After incubation and further washes, the counts present in the bound fraction can be determined and will be related directly to the amount of antigen added.
Descriptions of most conventional immunoassays may be found in Langan J. and Clapp J. 1981, Ligand Assay (Masson Publishing USA.); and in Methods in Enzymology, Vol. 70, part A, 1980 (Academic Press). Abbreviations used throughout are defined as follows:
IRMA=immunoradiometric assay
MC=monoclonal antibody
anti-IdAb=anti-idiotypic antibody
Ag=antigen or component to be determined
Ab1=antibody specific for Ag=first antibody =antibody 1
Ab2=antibody specific for the idiotype of Abl =antibody 2
BSA=bovine serum albumin
Pb44=44,000 dalton membrane protein fragment of Plasmodium berghei sporozites
2D12=a monoclonal anti-idiotypic antibody
3D11=a monoclonal antibody specific for Pb44
Ag=antigen or component to be determined
MC1=monoclonal antibody of Abl specificity
MC2=monoclonal antibody of Ab2 specificity
Ab-Ab complexes=complexes of antibody bound to antibody
MCIn=monoclonal antibody specific for insulin
KLH=Keyhole limpet hemocyanin
PBS=phosphate buffered saline
The present inventors have discovered a new immunoassay, which differs in one essential aspect from all other previously described immunoassays. The sensitivity and specificity of the classical radioimmunoassay or of IRMA depend primarily on the affinity of one antibody for an antigen. The present invention is based on two specific reactions which take place at or close to the antigen-combining site of one antibody and on the inhibiting effects that they have on each other. In the present invention, Ab1 binds antigen and Ab2 binds Ab1; that is two ligands can bind to Ab1, the antigen and Ab2. If Ab2 is labelled, the inhibitory effect of Ag on the reaction between Ab2 and Ab1 can be measured. It will be understood that the term antigen includes any substance to which an antibody can bind, specifically, including a hapten. The term assay includes both quantitative and qualitative tests.
The novel use of anti-IdAb in an immunoassay provides the following advantages: the assay can measure a single epitope on the antigen, and purification of the antigen is not required. No known immunoassay appears to combine these advantages. Furthermore, it is also convenient to measure, with the immunoassay of this invention, antibodies of a particular antigenic specificity and a particular idiotype.
The present invention is exemplified by the measurement of Pb44, a 44,000 dalton membrane protein found in the sporozites of Plasmodium berghei, a rodent malarial parasite. As such, the immunoassay of the present invention has the promise of providing a means of surveying and monitoring the infectivity of mosquito populations. It will be understood that the immunoassay of this invention can be applied to virtually any antigen, including proteins from Plasmodium species infective to humans, and other clinically significant antigens related to viral or bacterial infections, hormonal and enzymatic abnormalities and the like.
The present invention is exemplified by the isolation and cloning of a monoclonal anti-idiotypic antibody (2D12) specific for an idiotype of a monoclonal antibody (3D11) which specifically binds the Pb44 sporozoite surface antigen.
The isolation and characterization of Pb44 was described in U.S. application Ser. No. 234,096, filed Feb. 12, 1981, now U.S. Pat. No. 4,466,917. The isolation of the anti-idiotypic antibody, 2D12, was described by Potocnjak, P. et al., Science 215, 1637 (1982). A critical feature of the 2D12 anti-idiotypic antibody is its ability to compete with Pb44 for binding to the anti Pb44 antibody, 3D11. In the present invention, anti-idiotypic antibody can be used either to indirectly measure antigen or to directly measure antibody of a given idiotype. It may also be used for standardizing and monitoring vaccine preparations. It will be understood that the antibodies disclosed herein may be derived from any vertebrate species possessing an immune system, including humans, and such antibodies, including human antibodies, can be used in the immunoassays of the present invention.
Finally, the present invention pertains to a new method of screening monoclonal antibodies having anti-idiotypic specificity and having the capacity to compete with the antigen for binding to the idiotype. The immunoassay of this invention is applicable to the screening of protein fragments useful for developing synthetic or non-synthetic vaccines against malaria, or for vaccines against other diseases. In addition, the sensitivity of the immunoassay is sufficient to allow epidemiological surveys in the investigation of malaria and other vector-transmitted diseases, e.g. in the order of as few as 100 sporozites of P. berghei are readily detectable.